Bottom Line:
Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses.Nucleic acid staining revealed that they contain DNA or RNA.In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10(9) viruses per g of calcite.

ABSTRACTUsing three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake "La Salada de Chiprana", Spain, contain viruses with a diameter of 50-80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>10(10) viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10(9) viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as "nanobacteria" and that viruses may play a role in initiating calcification.

pone.0130552.g003: Biogenic calcite, extracted from the microbial mats, observed in scanning electron microscopy linked with energy dispersive X-ray spectrometry and in atomic force microscopy.A: SEM picture of a cross section of carbonate grains showing a surface area of about 50 × 40 µm cutting through two agglomerates of very small globules and large crystals. B and C: Maps of calcium (B) and magnesium (C) obtained by EDS analysis in the same area. D-E: High-resolution images showing the variety in size and shape of mineral grains and crystals that compose biogenic calcium carbonate agglutinates. The arrows point towards smaller agglomerates of roundish micro-grains. F: EDS spectrum of the (Ca,Mg)CO3 grain for the location indicated by the blue dots in A and D (note that the high Si peak, due to the glass slide, masks the Si present in the clay colloids which also comprise Al and K). G: Details of an agglomerate of roundish micro-grains showing micromorphologies of very small rounded globules. H, I: AFM (in intermittent contact mode) images of a 1 × 1 µm surface area showing the globular structure of the individual (Ca,Mg)CO3 micro-grains and presented as height (H) and phase (I) images. J, K: AFM images of a 4 × 4 µm surface area in the cross section in AFM showing topography (J) and phase (K) images at a magnification comparable to that used for SEM. White arrows point to viruses that have been studied at higher magnification, i.e., the viruses illustrated in Fig 4D–4E.

Mentions:
Left panel (A): Virus counts in the extracted water fraction (pore water and water soaked into the extracellular polymer matrix). Right panel (B): Virus attached to solid organic and mineral matter. (circles = without acidification, squares = after 10 min of acidification, see Methods). Description of the different layers: I: the top layer from 0 to 0.8 ± 0.2 mm depth, comprised dense populations of diatoms of the genera: Frustula, Cymbella, Denticula, Nitzschia and few bundles of C. chthonoplastes and filaments of CLB; II: layer from 0.8 ± 0.2 mm to 1.5 ± 0.2 mm depth that separated very well from the top layer and comprised lesser densities of diatoms with high quantities biogenic high-Mg calcite grains (cf. Fig 3); III: locally a very fine layer was observed at 1.5 ± 0.3 mm depth that was particularly enriched in biogenic high-Mg calcite embedded in an organic matrix; IV: A layer located below the high densities of biogenic calcium carbonate crystals occurred layer B from 1.5 ± 0.3 mm to 2.6 ± 0.3 mm depth which comprising bundles of C. chthonoplastes and filaments of CLB; V: layer from 2.6 ± 0.3 mm to 4 ± 0.3 mm depth corresponded to a transition zone where large amounts of mineral particles sand grains and biogenic calcite occurred intertwined with of C. chthonoplastes and filaments of CLB; VI: layer comprising black coloured sediment ranging from 4 ± 0.3 mm to 6 ± 0.5 mm depth.

pone.0130552.g003: Biogenic calcite, extracted from the microbial mats, observed in scanning electron microscopy linked with energy dispersive X-ray spectrometry and in atomic force microscopy.A: SEM picture of a cross section of carbonate grains showing a surface area of about 50 × 40 µm cutting through two agglomerates of very small globules and large crystals. B and C: Maps of calcium (B) and magnesium (C) obtained by EDS analysis in the same area. D-E: High-resolution images showing the variety in size and shape of mineral grains and crystals that compose biogenic calcium carbonate agglutinates. The arrows point towards smaller agglomerates of roundish micro-grains. F: EDS spectrum of the (Ca,Mg)CO3 grain for the location indicated by the blue dots in A and D (note that the high Si peak, due to the glass slide, masks the Si present in the clay colloids which also comprise Al and K). G: Details of an agglomerate of roundish micro-grains showing micromorphologies of very small rounded globules. H, I: AFM (in intermittent contact mode) images of a 1 × 1 µm surface area showing the globular structure of the individual (Ca,Mg)CO3 micro-grains and presented as height (H) and phase (I) images. J, K: AFM images of a 4 × 4 µm surface area in the cross section in AFM showing topography (J) and phase (K) images at a magnification comparable to that used for SEM. White arrows point to viruses that have been studied at higher magnification, i.e., the viruses illustrated in Fig 4D–4E.

Mentions:
Left panel (A): Virus counts in the extracted water fraction (pore water and water soaked into the extracellular polymer matrix). Right panel (B): Virus attached to solid organic and mineral matter. (circles = without acidification, squares = after 10 min of acidification, see Methods). Description of the different layers: I: the top layer from 0 to 0.8 ± 0.2 mm depth, comprised dense populations of diatoms of the genera: Frustula, Cymbella, Denticula, Nitzschia and few bundles of C. chthonoplastes and filaments of CLB; II: layer from 0.8 ± 0.2 mm to 1.5 ± 0.2 mm depth that separated very well from the top layer and comprised lesser densities of diatoms with high quantities biogenic high-Mg calcite grains (cf. Fig 3); III: locally a very fine layer was observed at 1.5 ± 0.3 mm depth that was particularly enriched in biogenic high-Mg calcite embedded in an organic matrix; IV: A layer located below the high densities of biogenic calcium carbonate crystals occurred layer B from 1.5 ± 0.3 mm to 2.6 ± 0.3 mm depth which comprising bundles of C. chthonoplastes and filaments of CLB; V: layer from 2.6 ± 0.3 mm to 4 ± 0.3 mm depth corresponded to a transition zone where large amounts of mineral particles sand grains and biogenic calcite occurred intertwined with of C. chthonoplastes and filaments of CLB; VI: layer comprising black coloured sediment ranging from 4 ± 0.3 mm to 6 ± 0.5 mm depth.

Bottom Line:
Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses.Nucleic acid staining revealed that they contain DNA or RNA.In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10(9) viruses per g of calcite.

ABSTRACTUsing three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake "La Salada de Chiprana", Spain, contain viruses with a diameter of 50-80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>10(10) viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10(9) viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as "nanobacteria" and that viruses may play a role in initiating calcification.